Animals
Male mice at 120 ± 2 days of age were used to avoid potential differences in Aldh1a1 neurons between sexes. Mice were bred and reared under the same conditions in accordance with our institutional guidelines and the Animal Care and Use Committee of the Animal Core Facility at Huazhong University of Science and Technology, Wuhan, China, and housed in groups of three to five mice/cage under a 12 h light-dark cycle, with lights on at 8:00 am, at a constant ambient temperature (21 ± 1 °C) and humidity (50 ± 5%). All behavioral tests were conducted during the light phase of the cycle. For touchscreen-based choice behavioral tests, the mice were maintained on a restricted diet and kept at 90% of their free-feeding body weight during behavioral testing. The animals were randomly allocated to different experimental conditions in this study. To target specifically to Aldh1a1 neurons, we generated Aldh1a1-CRE mice, in which CRE was expressed under the Aldh1a1 promoter (Supplementary Fig. 10a, b).
For the generation of Aldh1a1−/−-CRE mice, a P2A-CRE site was inserted downstream of exon 10. The deletion of exons 11–13 eliminated 100 amino acids (401–500) of the C-terminal, which is essential for enzyme function and stability of Aldh1a1 [18]. The vector design for the generation of Aldh1a1-CRE and Aldh1a1−/−-CRE is described in detail in Supplementary Fig. 10a, c. The absence of protein products was established by western blot analysis.
Amyloid model mice (APPswe/PSEN1dE9 mice, or AD mice) with a C57BL/6 genetic background were purchased from the Jackson Laboratory (Stock No.: 005864) and housed in the University animal center. In this study, male AD mice at 5 months old of age were used and identified as heterozygous by genotyping with the following primers:
5′- ATGGTAGAGTAAGCGAGAACACG-3’forward for mutant;
5′- GTGTGATCCATTCCATCAGC − 3’forward for wild type;
5′- GGATCTCTGAGGGGTCCAGT − 3′ reverse for common.
Cell labeling and monosynaptic tracing
To determine the synaptic targets of Aldh1a1 neurons, a high titer (0.1 μl, 8, × 1012 genomic particles/ml) of the CRE-recombination-dependent rAAV1/2-TH-DIO-TKGFP virus particles (helper virus) was stereotaxically injected into the VTA of Aldh1a1-CRE mice to express thymidine kinase (TK) in Aldh1a1 neurons. The coordinates of the stereotaxic virus injections were as follows: AP: −3.6, ML: ±0.7, DV: 4.0. The rAAV1/2-TH-DIO-TKGFP virus was generated by insertion of a double loxP-flanked inverted TK-2A-GFP sequence immediately downstream of the TH promoter in the rAAV vector, which were co-transfected with AAV helper1 and helper2 mixers (rAAV1/2) into HEK293 cells to generate a high titer of rAAV1/2-TH-DIO-TKGFP virus particles (3 × 1012 genomic particles/ml), as described previously [19]. The TH promoter was used because we wanted to express TKGFP specifically in Aldh1a1-expressing dopaminergic neurons. Twelve days after the injection of rAAV1/2-TH-DIO-TKGFP virus particles, 0.05 μl of a high titer (5 × 108 genomic particles/ml) of a genetically modified version of Herpes simplex virus type 1 strain 129 (H129ΔTK-tdT virus), in which TK was deleted, was then injected. The generation of H129ΔTK-tdT virus particles has been described previously [20, 21]. Seven days after the injection of H129ΔTK-tdT virus particles, the mice were sacrificed and fixed. Furthermore, 24 h after fixation, brain sections were imaged under a laser confocal microscope (Zeiss LSM 800, Zeiss). With the assistance of the helper virus, H129ΔTK-tdT transmits anterogradely through Aldh1a1 neurons to their postsynaptic neurons, as described previously [20,21,22].
To determine the presynaptic neurons of Aldh1a1 neurons, we expressed TVA/G proteins in Aldh1a1 neurons by injecting the rAAV1/2-TH-DIO-TVA/G-GFP virus into the VTA of Aldh1a1-CRE mice. A high titer (0.1 μl of 7 × 1010 genomic particles/ml) of the ΔG-rabies virus that encoded tdT (ΔRV) was applied to the same brain region. This injection caused specific labeling of Aldh1a1 neurons and their presynaptic L5PN. Construction and generation of rAAV1/2-TH-DIO-TVA/G-GFP and ΔRV virus particles have been described previously [19, 20, 22].
Whole-cell patch-clamp recordings with chemogenetics and optogenetics
To investigate synaptic transmission between Aldh1a1 neurons and EGNIS, we injected the rAAV1/2-TH-DIO-Gi-ChR2tdT, rAAV1/2-TH-DIO-TK, and H129ΔTK-FLP virus particles (BrainVTA Co., Ltd., China) into the VTA and the FLP-recombination-dependent rAAV1/2-CaMKIIα-fDIO-GFP virus into the intermediate lateral septum of Aldh1a1-CRE mice, resulting in the expression of Gi-ChR2tdT in Aldh1a1 neurons and GFP under the control of the CaMKIIα promoter in EGNIS. The coordinates of the stereotaxic virus injections were AP: 0.3, ML: ±0.5, DV: 3.0, in the intermediate lateral septum.
The slices were then prepared and transferred to a holding chamber that contained artificial cerebrospinal fluid (ACSF in mM: 124 NaCl, 3 KCl, 26 NaHCO3, 1.2 MgCl2, 1.25 NaH2PO42H2O, 10 C6H12O6, and 2 CaCl2 at pH 7.4, 305 mOsm) at 32 °C for 30 min. The temperature was maintained at 22 °C for 60 min. A slice was transferred to a recording chamber, which was continuously perfused with oxygenated ACSF (2 ml/min) at 22 °C. We performed whole-cell current-clamp recordings from GFP-expressing EGNIS in the slices, which were visualized under a fluorescent infrared-phase-contrast (IR-DIC) Axioskop 2FS upright microscope equipped with a Hamamatsu C2400-07E infrared camera, as described previously [19, 20, 22, 23]. Inhibitory postsynaptic currents (IPSCs) were evoked by the delivery of blue laser light onto axon fibers of ChR2-expressing Aldh1a1 neurons and inhibited by infusing 5 μM clozapine-N-oxide (CNO) onto the IS through a cannula. The membrane potentials of EGNIS were held at −70 mV. A high Cl− internal recording solution contained (in mM) 150 CsCl, 10 HEPES, 0.2 EGTA, 2 Mg-ATP, 0.3 guanosine triphosphate, and 0.1% biocytin, pH 7.4 (296 mOsm). The external ACSF solution contained 20 μM CNQX (TOCRIS, 0190) throughout the recordings. IPSCs were sensitive to 20 μM bicuculline (TOCRIS, 0130) GABAA receptor antagonist, showing a GABAA receptor-dependent synaptic response.
To record synaptic transmission between L5PN and Aldh1a1 neurons, we expressed Gi-ChR2tdT in L5PN and GFP in Aldh1a1 neurons. Specifically, we first injected rAAV1/2-TH-DIO-TVA/G and ΔRV-FLP virus particles (Taiting Biotechnology Co., Ltd., China) into the VTA of Aldh1a1-CRE mice to express FLP in L5PN. FLP-recombination-dependent rAAV1/2-fDIO-Gi-ChR2tdT virus was injected into layer five of the ventral medial prefrontal cortex (mPFC). This injection caused the expression of Gi-ChR2tdT in Aldh1a1 presynaptic L5PN. The coordinates of the stereotaxic virus injections were AP: 1.9, ML: ±0.5, DV: 3.0. Construction and generation of the rAAV virus particles have been described previously [19, 20, 22].
Next, we performed whole-cell current-clamp recordings of GFP-expressing Aldh1a1 neurons in the slices. Excitatory postsynaptic currents (EPSCs) were evoked by the delivery of blue laser light onto axon fibers of Gi-ChR2tdT-expressing L5PN at a holding potential of −70 mV and inhibited by infusion of 5 μM CNO into ACSF. The internal recording solutions consisted of (in mM) 140 potassium gluconate, 0.05 EGTA, 10 HEPES, 2 Mg-ATP, 0.2 GTP at pH 7.4 with 292 mOsm. The external ACSF solution contained GABAA receptor antagonists, including 20 μM bicuculline (TOCRIS, 0130). EPSCs were sensitive to 20 μM CNQX (TOCRIS, 0190), showing an AMPA receptor-dependent synaptic response. To record NMDA receptor-mediated EPSCs, which were sensitive to 100 μM DL-AP5 sodium salt (TOCRIS, 0105), the holding potential was switched from −70 mV to +60 mV.
Electrophysiology and optogenetics in vivo
We anesthetized mice with 6% chloral hydrate (0.06 ml/10 g; intraperitoneally) and planted the coated four tetrodes of twisted 17 μm HM-L with platinum-iridium (10% or 20% platinum, #: 100–167, California Fine Wire Company) with the coordinates of AP: −3.6, ML: ±0.7, DV: 3.5-4.0 in VTA, AP: 0.3, ML: ±0.5, DV: 2.7-3.2 in IS, and AP: 1.9, ML: ±0.5, DV: 2.8-3.3 in the mPFC, as described before [19, 20, 22]. We placed the tetrodes directly above the recording site and secured the driver to the skull using jeweler’s screws and dental cement. A jeweler screw was used as the ground electrode. We screened the cells and behaviors daily for each experimental procedure. During the screening procedures, we lowered the tetrodes slowly over several days in steps of 30 μm. For light stimulation of the ChR2-expressing neurons, we planted a bound 20 μm in diameter, unjacketed optical fiber (Inper Co., Ltd., China) in a tetrode-containing silicone tube (166 μm) into the VTA or layer 5b of the mPFC or the intermediate lateral septum. We validated the position of the optic fibers by electrolytic lesions after light stimulation. We applied 473 nm lasers (DPSS laser, Inper Co., Ltd., China) for light activation of targeting neurons or axon fibers. The laser power ranged from 1 to 5 mW/mm2 unless otherwise indicated.
Extracellular single units were recorded from Aldh1a1 and L5PNs. The mice were connected to the recording equipment via AC-coupled unity-gain operational amplifiers (Plexon, Dallas, TX, USA). The signals were amplified 4000- to 8000-fold, as described previously [19, 20, 22]. The spikes were recorded at the same time and isolated using a 250 Hz low-pass filter and a 250 Hz high-pass filter of the commercial software OmniPlex (Plexon). Spike sorting was performed offline using graphical cluster-sorting software (Offline Sorter, Plexon). To estimate the quality of the cluster separation, we calculated the isolation distance and L-ratio using Plexon SDK (www.plexon.com/software-downloads/SDK).
To isolate and analyze spike units from individual neuronal types, we calculated the valley-to-peak time and the half-width of the spikes. Spikes in Aldh1a1 neurons and L5PN were identified and distinguished from the cell types in the same brain regions based on the duration of the negative spike, the firing pattern (complex spikes), and the low average firing rate and validated via light activation of ChR2-expressing Aldh1a1 neurons and L5PN. The average firing rate was expressed as the total number of spikes divided by the total length of the recording period.
Microdialysis in vivo
We anesthetized mice with 6% chloral hydrate (0.06 ml/10 g) and implanted dialysis guide cannula for insertion of the CMA7 dialysis probe in the IS with the following coordinates of AP: 0.3, ML: ±0.5, DV: 3.0 and secured the cannula to the skull using jeweler’s screws and dental cement. Dialysis was performed 24 h after the probe implantation. The perfusion fluid was pumped through the dialysis probe at a rate of 2 μl/min. Samples were collected on ice containing 3.3 μl of dialysate buffer (0.1 M glacial acetic acid, 0.1 mM EDTA; HPLC grade reagent; and 0.12% oxidized l-glutathione, pH at 3.70). Then, 15 μl of the sample were placed in a polypropylene cryogenic vial with 5 μl of 50 nM DA-D4 in 1 mM HCl, 5 μl of 1 M NaHCO3, and 25 μl of freshly prepared 1% dansyl chloride solution in acetone. Samples were incubated at 65 °C for 10 min, chilled on ice for 2 min, and then stored in liquid nitrogen until quantification.
Blue laser light was delivered to ChR2-expressing Aldh1a1 neurons when a stable basal value was obtained. Glutamate, GABA, and dopamine were measured using high performance liquid chromatography with fluorescence detection (HPLC-FD, 150 × 4.6 mm, C18, 5 μm particle size column, Agilent Technologies, USA) coupled to a fluorescence detector (excitation wavelength: 340 nm, emission wavelength: 450 nm, RF-10AxL, Shimadzu Japan). The flow rate was 600 μl/min, the pressure was 463 bar, and the column temperature was set to 45 °C.
Open-field, object recognition
We measured motor activity within clear boxes (100 cm × 100 cm) and outfitted them with photo-beam detectors to monitor horizontal and vertical activity. Data were analyzed using the MED Associates Activity Monitor Data Analysis software. The mice were placed in the corner of the open-field apparatus and allowed to move freely. Behaviors including resting time (s), ambulatory time (s), vertical/rearing time (s), jump time (s), stereotypic time (s), and average velocity (cm/s) were assessed. The mice were not exposed to the chamber prior to the test. The data were recorded for each animal at 30 min intervals, as described previously [20].
To test the performance in the object recognition task, we subjected seven mice per group for two sessions of one trial each: acquisition and retrieval trials. During the acquisition trial, mice were placed in an arena containing two identical objects for 5 min. Mice that did not explore the objects for 20 s within the 5 min period were excluded from further experiments. We defined exploration as a mouse approaching its nose within 1 cm of an object. This approach was associated with looking, sniffing, or touching. The retrieval session was performed 2 h after the acquisition trial. In this trial, we replaced one of the objects presented in the first trial with a novel object. We then placed the mice back in the arena for 5 min and recorded the total time spent exploring each object. New objects were different in shape and color but were made of the same materials and had similar general dimensions. The objects and arenas were thoroughly cleaned with 70% ethanol between the trials. New objects and the positioning of new objects were counterbalanced in all experiments to avoid bias. Motor activity and time spent in active exploration of familiar or novel objects during the retrieval trial were calculated. The recognition index was expressed as the time spent exploring the novel object divided by the total time exploring both objects and multiplied by 100.
Delay of gratification touchscreen mouse model
We carried out touchscreen behavioral tasks in an automated touchscreen platform, comprising the Bussey-Saksida mouse touchscreen chamber (Lafayette Instrument, US) equipped with a house light, a reward port, holding a reward magazine with an infrared sensor for detection of a mouse entrance into the port, and a touch-sensitive monitor on the front side. All trials in the chamber were mouse initiated and independent of the experimenters. Testing consisted of pre-training, training, and testing sessions, and each behavioral group contained 9 mice.
In the pre-training session, mice were habituated to the apparatus and learned to nose poke to the stimuli presented in one of three windows, and then through several stages to associate the cue touching on the screen with the delivery of a reward (20 μl of chocolate milkshake, Bright Dairy co., Ltd., China) in the reward magazine as described previously [20, 22]. Once a mouse returned to the magazine and retrieved the reward, the magazine light was turned off, and an inter-trial interval of 20 s was initiated. Mice were subjected to the training session after 4 consecutive days (100 min per day, up to 60 trials). If a mouse failed to execute 60 trials within 60 min in the last day, this mouse was excluded from further experiments.
In the training session, mice were subjected to three types of reward learning tasks for 9 consecutive days, with 60 trials per day (one session per day, lasting up to 60 min), as shown in Supplementary Fig. 11. In the first type of learning task, the mice were trained to nose poke a cue symbol (flower) that was randomly displayed for 5 s in one of the three response windows on the touchscreen. Nose-poking this symbol resulted in a small immediate reward (SIR, 5 μl of chocolate milkshake at a 0-3 s delay). In the second type of learning task, mice were trained to nose poke a cue symbol (spider) that was randomly displayed for 5 s in one of the three response windows on the touchscreen. Nose-poking this symbol resulted in a large delayed reward (LDR, 20 μl of chocolate milkshake at a 6-9 s delay). In the third type of learning task, the mice were trained to nose poke a cue symbol (airplane) that was randomly displayed for 5 s in one of the three response windows on the touchscreen. Nose-poking this symbol resulted in a largest long delayed reward (LLR, 30 μl of chocolate milkshake at a 12-15 s delay). Each task consisted of 20 trials per day. After successful training (> 75% accuracy), the mice were subjected to probe trials. All groups of mice equally learned the behavioral performance throughout the training session.
In the probe trials, the mice were subjected to reward choice tasks, in which mice were required to freely choose between three cue symbols (airplane, spider, and flower) that were displayed for 5 s on the touchscreen, as demonstrated in Supplementary Movies 1-6 and Supplementary Fig. 11. Each symbol was associated with a specific reward (SIR, LDR, or LLR). The order of the symbols was randomized from trial to trial. The mice were allowed to poke only one of the three cue symbols in each trial. Each mouse performed 60 trials per day (one session per day, lasting up to 60 min) for 9 consecutive days. All data presented in this study were derived from probe trials.
Definitions: The time from cue presentation on the touchscreen to nose-poking was defined as the reaction time (R.T). Failure to nose-poking within 5 s was defined as an omitted trial. The time from nose-poking to triggering the infrared of the reward port was defined as the reward-collection delay (RCD). The correct collection of a contingency reward (RCD within the reward delay of SIR, LDR, or LLR) after nose-poking was defined as a correct trial. An incorrect collection of the cue reward (either before or after the reward delay of SIR, LDR, or LLR) was defined as an incorrect trial. A warning white noise with 1 s was instantly given to the mouse after an omitted trial or an incorrect trial. The correct score (C.S) was defined as the percentage of the number of correct trials versus the total number of trials. The trials% was defined as the percentage of the number of correct trials versus the total number of trials on each day of the probe trials. Accuracy was defined as the percentage of the number of correct trials versus the total number of trials on each day of SIR, LDR, and LLR separately.
Delay of gratification T-maze tests
A modified version of an automatic T-maze apparatus that was matte gray in color and consisted of three arms was used. There was one starting arm and two goal arms (Probecare Scientific, Co., Ltd., China) equipped with a starting box at the end of a start arm and a reward (sugar pellets, 14 mg, Bio-Serv) port holding a reward box with an infrared sensor detecting a mouse entrance into the port in each goal box. Two sliding doors were located at the entrance of each goal arm and the reward box for the restriction of a mouse in this goal arm during the delay period after making a choice. The behavioral testing consisted of habituation, training, and testing sessions, and each behavioral group contained 11 mice.
During habituation, the mice were habituated to the T-maze for a total of 5 days. On day one, the sugar pellets were scattered throughout the maze, and on days two and three, the sugar pellets were placed along the two-goal arms, and on days four and five, the sugar pellets were located at the two-goal boxes. The mice were placed in the start box of the maze and allowed to explore the maze for 10 min each day.
In training sessions, mice were allowed to visit one arm only at a given trial: either a large reward arm (LRA with three sugar pellets after a delay of 0-3 s) or a small reward arm (SRA with one sugar pellet after a delay of 0-3 s). After the mouse entered the goal arm, the sliding doors were closed until the delay was completed. Each mouse performed 50 trials (25 LRAs + 25 SIRs) per day (one session per day, lasting up to 60 min) for 5 consecutive days. After successful training, the mice were subjected to testing sessions.
In the testing sessions, the mice were allowed to visit the LRA with three sugar pellets after a delay of 0-3 s or 6-9 s) or SRA (with one sugar pellet only after a delay of 0-3 s). Each mouse performed 50 trials (LRA with three sugar pellets after a delay of 0-3 s in the 1-25 trials and 6-9 s in the 26-50 trials) per day (one session per day, lasting up to 60 min) for 5 consecutive days. To prevent the effects of spatial discrimination, the LRA location was counterbalanced with 50% mice on the left and the other 50% mice on the right. The percentage of LRA visits (LRA %) was defined as the percentage of LRA visiting trials versus the total number of trials on days one, three, and five of the testing sessions.
Western blots
We expressed GFP in Aldh1a1 neurons and isolated GFP-expressing Aldh1a1 neurons from the VTA of adult mice. In brief, 12 days after the injection of the rAAV1/2-TH-DIO-GFP virus into the VTA of Aldh1a1-CRE mice, the slices were prepared and digested in buffer containing 10 mM Tris-Cl (pH 7.6), 50 mM NaF, 1 mM Na3VO4, 1 mM edetic acid, 1 mM benzamidine, 1 mM PMSF, 1 mg/10 ml papain, and a mixture of aprotinin, leupeptin, and pepstatin A (10 μg/ml each) for 30 min. Suspended GFP-expressing Aldh1a1 neurons were automatically isolated using an S3e Cell Sorter (Bio-Rad), homogenized, and diluted with a buffer containing 200 mM Tris-Cl (pH 7.6), 8% SDS, and 40% glycerol. The protein concentration was determined using a BCA kit (Pierce, Rockford, IL, USA). The final concentrations of 10% β-mercaptoethanol and 0.05% bromophenol blue were added, and the samples were boiled for 10 min in a water bath. The proteins in the extracts were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The blots were scanned using an infrared imaging system (Odyssey, LI-COR). The blots were incubated with the following antibodies: goat anti-C-terminal-Aldh1a1 (1: 2000, Sigma-Aldrich, SAB2500058) and rabbit anti-α-tubulin (1:2000, Abcam, ab18251), and the band densities were quantitatively analyzed using Kodak Digital Science 1D software (Eastman Kodak, New Haven, CT), as described previously [22, 23]. The full-blot images can be found in the additional file (Original blots).
Immunohistochemistry
The mice were sacrificed by intraperitoneal injection of an overdose of chloral hydrate and were transcardially perfused with 100 mL saline (0.9% w/v NaCl), followed by 4% paraformaldehyde (PFA). The brains were removed and post-fixed in 4% PFA. Sagittal or coronal sections (30 μm) were sliced (Leica Microsystems, Wetzlar, Germany). Immunohistochemistry was performed on free-floating brain sections, as described previously [22,23,24]. In brief, staining was performed on 30 μm free-floating coronal sections and blocked in 3% normal donkey serum (room temperature for 1 h). For goat antibodies, donkey serum was used. The sections were then incubated in 50 mM Tris-HCl buffer containing 3% donkey serum and 0.3% Triton X-100 with one of the following primary antibodies: rabbit anti-Aldh1a1 (1: 1000, Abcam, ab52492), mouse anti-CaMKIIα (1: 3000, Abcam, ab22609), goat anti-CHAT (1:2000, Millipore, AB144P), mouse anti-GAD67 (1: 1000, Millipore, MAB5406), rabbit anti-TH (1: 1000, Abcam, ab112), and rat anti-CTIP2 (1: 500, Abcam, ab18465) for 24 h. Sections were rinsed with Tris-HCl buffer containing 3% donkey serum and 0.3% Triton X-100 and reacted with Alexa Fluor 488 donkey anti-rabbit, Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 546 donkey anti-rabbit, Alexa Fluor 488 donkey anti-goat, Alexa Fluor 546 donkey anti-mouse, Alexa Fluor 488 donkey anti-rat at room temperature for 1 h. The sections were rinsed, dried, and cover-slipped with a fluorescence mounting medium. The control sections were processed by omitting the primary antisera. Single or double labeling was viewed and imaged with a confocal laser-scanning microscope (Zeiss LSM800 Examiner Z1) and analyzed with a three-dimensional constructor (Image-Pro Plus software). A confocal series of images were taken at 0.5 μm intervals through the region of interest, and optical stacks of 6-12 images were produced for the figures. We quantified the absolute numbers of single, double, or triple labeled cells by sampling every section (image stacks) from the experimental animals, as described previously [19, 20, 22]. For cell counting, the experimenters coded all slides from the experiments before quantitative analysis. Quantification was performed by other experimenters who were unaware of the experimental conditions and treatments, as described previously [19, 20, 23].
Statistical analysis
All values in the text and figure legends are represented as the mean ± SEM. Unpaired two-tailed Student’s t-tests (t-test) and one-way analysis of variance (ANOVA) and post hoc Bonferroni’s following a two-way ANOVA (BF ANOVA) were used when assumptions of normality and equal variance (F test) were met (Supplementary Table 1). Statistical significance was accepted at a p-value of < 0.05. Power calculations were performed using G*power software version 3.1.9.2 (IDRE Research Technology Group, Los Angeles, USA). The group sizes were estimated based on recent studies and designed to provide at least 80% power with the following parameters: probability of type I error (α) = 0.05, conservative effect size of 0.25, and three to eight treatment groups with multiple measurements obtained per replicate.